Mixture of HMOs

ABSTRACT

A synthetic mixture of human milk oligosaccharides (HMOs) consisting essentially of lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), 2′-fucosyllactose (2′FL), 3′-O-sialyllactose (3′-SL), 6′-O-sialyllactose (6′-SL) either difucosyllactose (DFL) or 3-fucosyllactose (3-FL), preferably DFL, and optionally lactose. Said synthetic composition is useful for: treating or preventing viral and/or bacterial infection in a non-infant human; modulating the microbiota of a non-infant human; and/or improving the cognitive function of a non-infant human and as a pharmaceutical or nutritional composition.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a national stage entry pursuant to 35 U.S.C. § 371of International Patent Application No. PCT/IB2016/057675, filed on Dec.15, 2016, which claims priority to European Patent Application No.15200067.5, filed. Dec. 15, 2015, and European Patent Application No.16154144.6, filed Feb. 3, 2016, the contents of all of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to synthetic mixtures of human milkoligosaccharides (“HMOs”), particularly of LNnT, LNT, 2′-FL, 3′-SL,6′-SL and either DFL or 3-FL and applications of the mixtures in humanhealth.

BACKGROUND OF THE INVENTION

HMOs have become the subject of much interest in recent years due totheir roles in numerous biological processes occurring in the humanorganism. Mammalian milk contains at least 130 of these complexoligosaccharides (Urashima et al, Milk Oligosaccharides, Nova BiomedicalBooks, New York, 2011, ISBN: 978-1-61122-831-1).

Previously, the only source of HMOs had been mammalian milk whichcontains mostly water, together with 55-70 g/l lactose, 24-59 g/llipids, ca. 13 g/l proteins, 5-15 g/l HMOs and ca. 1.5 g/l minerals.

However, several processes for synthesizing HMOs have been developed inrecent years due to their roles in numerous human biological processes.In this regard, processes have been developed for producing HMOs bymicrobial fermentations, enzymatic processes, chemical syntheses, orcombinations of these technologies. For example, by chemical processes,Galpβ1-4GlcNAcpβ1-3Galpβ1-4Glc or lacto-N-neotetraose (“LNnT”) can bemade as described in WO 2011/100980 and WO 2013/044928;Galpβ1-3GlcNAcpβ1-3Galpβ1-4Glc or lacto-N-tetraose (“LNT”) can besynthesized as described in WO 2012/155916 and WO 2013/044928;6′-O-sialyllactose (“6′-SL) can be synthesized as described in WO2011/100979; a mixture of LNT and LNnT can be made as described in WO2013/091660; 2′-O-fucosyllactose (“2′-FL”) can be made as described inWO 2010/115934 and WO 2010/115935; 3-fucosyllactose (“3-FL”) can be madeas described in WO 2013/139344; and 6′-SL can be made as described in WO2010/100979. As examples of biotechnological processes, WO 01/04341 andWO 2007/101862 describe how to make core human milk oligosaccharidesoptionally substituted by fucose or sialic acid, including LNnT, 6′-SLand 3′-O-sialyllactose (“3′-SL”) using genetically modified E. coli; andWO 2015/032412 describes making 2′-FL and difucosyllactose orFuc(α1-2)Gal(β1-4)[Fuc(α1-3)]Glc (“DFL”) using genetically modified E.coli. As an example of enzymatic processes, sialylated oligosaccharidescan be made as described in EP-A-577580.

Efforts have also been made to develop processes for synthesizingenzymatically mixtures of HMO oligosaccharides, without having tosynthesize all of the component oligosaccharides of the mixture asdescribed in WO 2012/156897 and WO 2012/156898. Such processes haveprovided reaction mixtures containing a plurality of differentoligosaccharides.

Evidence is accumulating that the resident community of microbes, calledthe microbiota, in the human digestive tract plays a major role inhealth and disease. When the composition of the intestinal microbiota isthrown off balance, the human host can suffer consequences. Recentresearch has implicated intestinal microbiota imbalances in individualdisorders as diverse as cancer, obesity, inflammatory bowel disease,psoriasis, asthma, and possibly even autism. Individual non-digestiblefibres, including HMOs, are believed to beneficially modulate themicrobiota, and they are of increasing interest for treating one or moreof such disorders. However, many digestible fibres non-specificallymodulate the microbiota while others are not able to providesufficiently broad, but specific, modulation.

Therefore, there has been a need to specifically modulate themicrobiota, so as to address individual disorders in different ways andalso to address simultaneously multiple disorders. In particular, therehas been a need for a composition that can be used for, inter alia,treating and/or preventing bacterial and viral infections, particularlyin the intestinal and respiratory tracts, improving cognitive functionand/or increasing the efficacy of anticancer agents against tumors.

SUMMARY OF THE INVENTION

A first aspect of this invention relates to a synthetic mixture of HMOsconsisting essentially of LNnT, LNT, 2′-FL, 3′-SL, 6′-SL and either DFLor 3-FL, preferably DFL. This mixture can optionally include lactose.The mixture of HMOs preferably consists essentially of:

-   -   i. about 55 wt % to about 75 wt % of 2′-FL, more preferably        about 60 wt % to about 70 wt % of 2′-FL;    -   ii. about 2 wt % to about 10 wt % of LNnT, more preferably about        3 wt % to about 7 wt % of LNnT;    -   iii. about 10 wt % to about 20 wt % of LNT, more preferably        about 12 wt % to about 18 wt % of LNT;    -   iv. about 1 wt % to about 15 wt % of DFL or 3-FL, more        preferably about 1 wt % to about 10 wt %, even more preferably        about 2 wt % to about 10 wt % of DFL or 3-FL such as e.g. about        2 wt % to about 8 wt %;    -   v. about 1 wt % to about 10 wt % of 3′-SL, more preferably about        2 wt % to about 8 wt % of 3′-SL; and    -   vi. about 1 wt % to about 15 wt % of 6′-SL, preferably about 5        wt % to about 15 wt % of 6′-SL, more preferably about 7 wt % to        about 13 wt % of 6′-SL.

A second aspect of the invention relates to a nutritional orpharmaceutical composition comprising a synthetic mixture of HMOsaccording to the first aspect of the invention.

A third aspect of this invention relates to a synthetic mixture of HMOsor a composition comprising a synthetic mixture of HMOs for use in: i)preventing and/or treating viral and/or bacterial infections in anon-infant human; ii) specifically modulating the indigenous microbiotaof a non-infant human; and/or iii) improving the cognitive function of anon-infant human. The synthetic mixture of HMOs consists essentially ofLNnT, LNT, 2′-FL, 3′-SL, 6′-SL, either DFL or 3-FL, preferably DFL, andoptionally lactose, as described above. The synthetic HMO mixture or thenutritional or pharmaceutical composition comprising it contains aplurality of different HMOs with novel combinations of properties andbiological activities. The composition is especially useful againstviral and bacterial, intestinal infections through specific modulationof the intestinal microbiota by an increase in Bifidobacterium,modulation of intestinal binding of viruses and pathogenic bacteria tointestinal epithelial cells, and improvement of intestinal barrierfunction. The composition is also especially useful against viral andbacterial, respiratory tract infections by inhibiting pathogen bindingto human epithelial cells.

A fourth aspect of this invention relates to a method of modulating theindigenous microbiota of a non-infant human to increase the abundance ofBifidobacterium in order to increase the efficacy of anticancer agentsagainst tumors in a non-infant human patient. The method involvesadministering to the non-infant human a synthetic mixture of HMOsaccording to the first aspect of this invention or a nutritional orpharmaceutical composition according to the second aspect of thisinvention, as described above. Bifidobacterium can act as an immunebooster, hence the increase in its abundance can strengthen a cancerpatient's response to an anticancer agent. This property makes themixture suitable as an aid in cancer therapy.

A fifth aspect of this invention relates to a method of modulating theindigenous intestinal microbiota of a non-infant human to increaseBifidobacterium and/or Barnesiella abundance and also to reduce theabundance of Ruminococcus gnavus. The method involves administering tothe non-infant human a synthetic mixture of HMOs according to the firstaspect of this invention or a nutritional or pharmaceutical compositionaccording to the second aspect of this invention, as described above.The increased Bifidobacterium and/or Barnesiella abundance and reducedabundance of Ruminococcus gnavus render the non-infant human'sintestinal milieu less prone to inflammation and provide improvedintestinal barrier function. These effects can prevent and/or treatconditions such as inflammatory bowel disease, irritable bowel syndrome,and other conditions associated with inflammation and impaired gutbarrier function.

A sixth aspect of this invention relates to a method of modulating theindigenous microbiota of a non-infant human to increase Bifidobacteriumabundance and to at least maintain the abundance of Faecalibacterium.The method involves administering to the non-infant human a syntheticmixture of HMOs according to the first aspect of this invention or anutritional or pharmaceutical composition according to the second aspectof this invention, as described above. Increasing the Bifidobacteriumabundance and at least maintaining the abundance of Faecalibacteriumrender the non-infant human's intestinal milieu less prone toinflammation and provide improved intestinal barrier function.Preferably the abundance of Ruminococcus gnavus is reduced. Theseeffects can prevent and/or treat conditions such as inflammatory boweldisease, irritable bowel syndrome, and other conditions associated withinflammation and impaired gut barrier function.

In both the fifth and sixth aspects of the invention, the abundance ofProteobacteria is also preferably reduced.

A seventh aspect of this invention relates to a method of preventing ortreating viral and/or bacterial infections in a non-infant human,especially intestinal infections and infections of the respiratorytract. The method comprises administering, to the non-infant human, asynthetic mixture of HMOs according to the first aspect of thisinvention or a nutritional or pharmaceutical composition according tothe second aspect of this invention, as described above.

An eighth aspect of this invention relates to a method of improving thecognitive function of a non-infant human. The method comprisesadministering, to the non-infant human, a synthetic mixture of HMOsaccording to the first aspect of this invention or a nutritional orpharmaceutical composition according to the second aspect of thisinvention, as described above.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with this invention, the following terms preferably havethe following meanings:

“Non-infant human” or “non-infant” preferably means a human of 3 yearsof age and older. Accordingly, a non-infant human is a human of any ageabove 3 years old, e.g. it can be a child, a teenager, an adult or anelderly person.

“Synthetic mixture of HMOs” means a mixture which is artificiallyprepared and preferably means a mixture in which at least one HMO isproduced ex vivo chemically and/or biologically, e.g. by means ofchemical reaction, enzymatic reaction or recombinantly. More preferably,all of the HMOs in the mixture are produced ex vivo chemically and/orbiologically. A synthetic mixture of the invention is not identical witha naturally occurring mixture.

“Microbiota”, “microflora” and “microbiome” mean a community of livingmicroorganisms that typically inhabits a bodily organ or part,particularly the gastro-intestinal organs of non-infant humans. The mostdominant members of the gastrointestinal microbiota includemicroorganisms of the phyla of Firmicutes, Bacteroidetes,Actinobacteria, Proteobacteria, Synergistetes, Verrucomicrobia,Fusobacteria, and Euryarchaeota; at genus level Bacteroides,Faecalibacterium, Bifidobacterium, Roseburia, Alistipes, Collinsella,Blautia, Coprococcus, Ruminococcus, Eubacterium and Dorea; at specieslevel Bacteroides uniformis, Alistipes putredinis, Parabacteroidesmerdae, Ruminococcus bromii, Dorea longicatena, Bacteroides caccae,Bacteroides thetaiotaomicron, Eubacterium hallii, Ruminococcus torques,Faecalibacterium prausnitzii, Ruminococcus lactaris, Collinsellaaerofaciens, Dorea formicigenerans, Bacteroides vulgatus and Roseburiaintestinalis. The gastrointestinal microbiota includes themucosa-associated microbiota, which is located in or attached to themucus layer covering the epithelium of the gastrointestinal tract, andluminal-associated microbiota, which is found in the lumen of thegastrointestinal tract.

“Modulating of microbiota” means exerting a modifying or controllinginfluence on microbiota, in particular an influence leading to anincrease in the indigenous intestinal abundance of Bifidobacterium,Barnesiella and/or Faecalibacterium, and reduction of the intestinalabundance of Ruminococcus gnavus and/or Proteobacteria.

“Proteobacteria” are a phylum of Gram-negative bacteria and include awide variety of pathogenic bacteria, such as Escherichia, Salmonella,Vibrio, Helicobacter, Yersinia and many other notable genera.

“Therapy” means treatment given or action taken to reduce or eliminatesymptoms of a disease or pathological condition.

“Preventive treatment” or “prevention” in the present context means atreatment given or an action taken to diminish the risk of the onset orrecurrence of a disease.

“Viral infection” in the present context means an infection of thestomach and intestines, or any other parts of the gastro-intestinaltract, by a virus often resulting in inflammatory response, commonlyknown as the stomach flu. The symptoms may include one or more of thefollowing: watery diarrhoea, nausea and vomiting, headache, muscleaches, joint aches fever, chills, sweating, clammy skin, abdominalcramps and pain, loss of appetite, weight loss. Examples of viralgastroinfections include, but not limited to, viral gastroinfectionscaused by Rotavirus or Norovirus.

“Bacterial infection” in the present context means an infection of thestomach and intestines, or any other parts of the gastro-intestinaltract, by a pathogenic bacteria, commonly known as bacterial entheritis.The symptoms may include one or more of the following: loss of appetite,nausea and vomiting, diarrhoea, abdominal pains and cramps, blood in thestool, fever. Examples of bacterial infections include, but not limitedto, gastroinfections by pathogenic strains of Yersinia, Staphylococcus,Shigella, Salmonella, Campylobacter and E. Coli.

“Infections of the respiratory tract” refers to any viral and/orbacterial infections of upper and lower respiratory tract, i.e.infections of the sinuses, throat, airways or lungs.

“Cancer” preferably refers to a neoplasm; for example of any part of oneor more of the following: respiratory, gastro-intestinal, urinary tractsor liver.

“Oral administration” means any conventional form for the delivery of acomposition through the mouth. Accordingly, oral administration is aform of enteral administration.

“Cognitive function” refers to cerebral activities that lead toknowledge, including all means and mechanisms of acquiring information.Cognitive functions encompass reasoning, memory, attention, and languageand lead directly to the attainment of information and, thus, knowledge.

In accordance with this invention, it has been surprisingly discoveredthat a synthetic HMO mixture consisting essentially of LNnT, LNT, 2′-FL,3′-SL, 6′-SL and either DFL or 3-FL, preferably DFL, and optionallylactose, can provide an anti-infective composition for preventing ortreating, bacterial or viral infections through specific modulation ofthe intestinal microbiota, binding of viruses, reduction in pathogenictranslocation and improvement of intestinal barrier function in anon-infant human. Further, the HMO mixture of this invention acts as adecoy receptor and binds to rotaviruses to prevent the rotaviruses fromadhering to human intestinal cells. These properties, coupled with animprovement in intestinal barrier function, make the HMO mixturesuitable for preventing and treating intestinal infections.

It has also been found that the HMO mixture of this invention canincrease the brain ganglioside and glycoprotein sialic acidconcentrations, and enhance hippocampal long term potentiation throughthe vagus nerve, leading to increased synaptogenesis andneurodevelopment in a non-infant human. This makes the HMO mixturesuitable for administration to non-infant humans to improve theircognitive function.

The HMO mixture of this invention can also:

-   -   i) increase the indigenous intestinal abundance of        Bifidobacterium, and    -   ii) increase the intestinal abundance of Barnesiella and/or at        least maintain the intestinal abundance of Faecalibacterium, and    -   iii) reduce the intestinal abundance of Ruminococcus gnavus        and/or Proteobacteria

in non-infant humans.

These effects can render an intestinal milieu less prone toinflammation. Coupled with an improvement in intestinal barrierfunction, these effects of the HMO mixture can prevent and/or treatconditions such as inflammatory bowel disease, irritable bowel syndrome,and other conditions associated with inflammation and impaired barrierfunction.

Surprisingly, the increase of bifidobacteria in the gut, induced by theHMO mixture of this invention, increases the efficacy of anticanceragents against tumors. The bifidobacteria act as immune helpers,strengthening a cancer patient's response to an anticancer agent. Thisproperty makes the HMO mixture suitable as an aid in cancer therapy.

Preferably the HMO mixture of this invention contains: i) about 55 wt %to about 75 wt % of 2′-FL, more preferably about 60 wt % to about 70 wt%; ii) about 2 wt % to about 10 wt % of LNnT, more preferably about 3 wt% to about 7 wt %; iii) about 10 wt % to about 20 wt % of LNT, morepreferably about 12 wt % to about 18 wt %; iv) about 1 wt % to about 15wt % of DFL or 3-FL, preferably about 1 wt % to about 10 wt %, even morepreferably about 2 wt % to about 10 wt %; v) about 1 wt % to about 10 wt% of 3′-SL, more preferably about 2 wt % to about 8 wt %; and vi) about1 wt % to about 15 wt % of 6′-SL, preferably about 5 wt % to about 15 wt%, more preferably about 7 wt % to about 13 wt %. The HMO mixture ofthis invention can also contain lactose, but it is not considered anactive ingredient of the mixture.

The synthetic HMO mixture can be administered to a non-infant human inany suitable form such as a nutritional or pharmaceutical composition,for example, a unit dosage form (for example, a tablet, a capsule, asachet of powder, etc.).

The nutritional or pharmaceutical composition consists of the syntheticmixture of HMOs consisting essentially of LNnT, LNT, 2′-FL, 3′-SL,6′-SL, either DFL or 3-FL, and optionally lactose, and suitableexcipients. The nutritional or pharmaceutical composition does notcontain any other HMOs than those mentioned above.

In one embodiment, the HMO mixture of this invention can be in the formof a nutritional composition. For example, the nutritional compositioncan be a food composition, a rehydration solution, or a dietarymaintenance or supplement for elderly individuals or immunocompromisedindividuals. The nutritional composition can contain sources of protein,lipids and/or digestible carbohydrates and can be in powdered or liquidforms. The composition can be designed to be the sole source ofnutrition or a nutritional supplement.

Suitable protein sources include milk proteins, soy protein, riceprotein, pea protein and oat protein, or mixtures thereof. Milk proteinscan be in the form of milk protein concentrates, milk protein isolates,whey protein or casein, or mixtures of both. The protein can be wholeprotein or hydrolysed protein, either partially hydrolysed orextensively hydrolysed. Hydrolysed protein offers the advantage ofeasier digestion which can be important for non-infants with inflamed GItracts. The protein can also be provided in the form of free aminoacids. The protein can comprise about 5% to about 30% of the energy ofthe nutritional composition, normally about 10% to 20%.

The protein source can be a source of glutamine, threonine, cysteine,serine, proline, or a combination of these amino acids. The glutaminesource can be a glutamine dipeptide and/or a glutamine enriched protein.Glutamine can be included due to the use of glutamine by enterocytes asan energy source. Threonine, serine and proline are important aminoacids for the production of mucin. Mucin coats the GI tract and canimprove mucosal healing. Cysteine is a major precursor of glutathione,which is key for the antioxidant defences of the body.

Suitable digestible carbohydrates include maltodextrin, hydrolysed ormodified starch or corn starch, glucose polymers, corn syrup, corn syrupsolids, high fructose corn syrup, rice-derived carbohydrates,pea-derived carbohydrates, potato-derived carbohydrates, tapioca,sucrose, glucose, fructose, sucrose, lactose, honey, sugar alcohols(e.g., maltitol, erythritol, sorbitol), or mixtures thereof. Preferablythe composition is free from added lactose. Generally digestiblecarbohydrates provide about 35% to about 55% of the energy of thenutritional composition. A particularly suitable digestible carbohydrateis a low dextrose equivalent (DE) maltodextrin.

Suitable lipids include medium chain triglycerides (MCT) and long chaintriglycerides (LCT). Preferably the lipid is a mixture of MCTs and LCTs.For example, MCTs can comprise about 30% to about 70% by weight of thelipids, more specifically about 50% to about 60% by weight. MCTs offerthe advantage of easier digestion which can be important for non-infantswith inflamed GI tracts. Generally, the lipids provide about 35% toabout 50% of the energy of the nutritional composition. The lipids cancontain essential fatty acids (omega-3 and omega-6 fatty acids).Preferably these polyunsaturated fatty acids provide less than about 30%of total energy of the lipid source. Decreasing the levels of thesepolyunsaturated fatty acids is believed to decrease sensitivity toperoxidation; which can be beneficial for non-infants havinginflammatory conditions.

Suitable sources of long chain triglycerides are rapeseed oil, sunflowerseed oil, palm oil, soy oil, milk fat, corn oil, high oleic oils, andsoy lecithin. Fractionated coconut oils are a suitable source of mediumchain triglycerides. The lipid profile of the nutritional composition ispreferably designed to have a polyunsaturated fatty acid omega-6 (n-6)to omega-3 (n-3) ratio of about 4:1 to about 10:1. For example, the n-6to n-3 fatty acid ratio can be about 6:1 to about 9:1.

The nutritional composition may also include vitamins and minerals. Ifthe nutritional composition is intended to be a sole source ofnutrition, it preferably includes a complete vitamin and mineralprofile. Examples of vitamins include vitamins A, B-complex (such as B1,B2, B6 and B12), C, D, E and K, niacin and acid vitamins such aspantothenic acid, folic acid and biotin. Examples of minerals includecalcium, iron, zinc, magnesium, iodine, copper, phosphorus, manganese,potassium, chromium, molybdenum, selenium, nickel, tin, silicon,vanadium and boron.

The nutritional composition can also include a carotenoid such aslutein, lycopene, zeaxanthin, and beta-carotene. The total amount ofcarotenoid included can vary from about 0.001 μg/ml to about 10 μg/ml.Lutein can be included in an amount of from about 0.001 μg/ml to about10 μg/ml, preferably from about 0.044 μg/ml to about 5 μg/ml of lutein.Lycopene can be included in an amount from about 0.001 μg/ml to about 10μg/ml, preferably about 0.0185 μg/ml to about 5 μg/ml of lycopene.Beta-carotene can comprise from about 0.001 μg/ml to about 10 mg/ml, forexample about 0.034 μg/ml to about 5 μg/ml of beta-carotene.

The nutritional composition preferably also contains reducedconcentrations of sodium; for example, from about 300 mg/l to about 400mg/l. The remaining electrolytes can be present in concentrations set tomeet needs without providing an undue renal solute burden on kidneyfunction. For example, potassium is preferably present in a range ofabout 1180 to about 1300 mg/l; and chloride is preferably present in arange of about 680 to about 800 mg/l.

The nutritional composition can also contain various other conventionalingredients such as preservatives, emulsifying agents, thickeningagents, buffers, fibres and prebiotics (e.g. fructooligosaccharides,galactooligosaccharides), probiotics (e.g. B. animalis subsp. lactisBB-12, B. lactis HN019, B. lactis Bi07, B. infantis ATCC 15697, L.rhamnosus GG, L. rhamnosus HNOOI, L. acidophilus LA-5, L. acidophilusNCFM, L. fermentum CECT5716, B. longum BB536, B. longum AH1205, B.longum AH1206, B. breve M-16V, L. reuteri ATCC 55730, L. reuteri ATCCPTA-6485, L. reuteri DSM 17938), antioxidant/anti-inflammatory compoundsincluding tocopherols, caroteinoids, ascorbate/vitamin C, ascorbylpalmitate, polyphenols, glutathione, and superoxide dismutase (melon),other bioactive factors (e.g. growth hormones, cytokines, TFG-β),colorants, flavours, and stabilisers, lubricants, and so forth.

The nutritional composition can be formulated as a soluble powder, aliquid concentrate, or a ready-to-use formulation. The composition canbe fed to a human in need via a nasogastric tube or orally. Variousflavours, fibres and other additives can also be present.

The nutritional compositions can be prepared by any commonly usedmanufacturing techniques for preparing nutritional compositions in solidor liquid form. For example, the composition can be prepared bycombining various feed solutions. A protein-in-fat feed solution can beprepared by heating and mixing the lipid source and then adding anemulsifier (e.g. lecithin), fat soluble vitamins, and at least a portionof the protein source while heating and stirring. A carbohydrate feedsolution is then prepared by adding minerals, trace and ultra traceminerals, thickening or suspending agents to water while heating andstirring. The resulting solution is held for 10 minutes with continuedheat and agitation before adding carbohydrates (e.g. the HMOs anddigestible carbohydrate sources). The resulting feed solutions are thenblended together while heating and agitating and the pH adjusted to6.6-7.0, after which the composition is subjected to high-temperatureshort-time processing during which the composition is heat treated,emulsified and homogenized, and then allowed to cool. Water solublevitamins and ascorbic acid are added, the pH is adjusted to the desiredrange if necessary, flavours are added, and water is added to achievethe desired total solid level.

For a liquid product, the resulting solution can then be asepticallypacked to form an aseptically packaged nutritional composition. In thisform, the nutritional composition can be in ready-to-feed orconcentrated liquid form. Alternatively, the composition can bespray-dried and processed and packaged as a reconstitutable powder.

When the nutritional product is a ready-to-feed nutritional liquid, itmay be preferred that the total concentration of HMOs in the liquid, byweight of the liquid, is from about 0.0001% to about 2.0%, includingfrom about 0.001% to about 1.5%, including from about 0.01% to about1.0%. When the nutritional product is a concentrated nutritional liquid,it may be preferred that the total concentration of HMOs in the liquid,by weight of the liquid, is from about 0.0002% to about 4.0%, includingfrom about 0.002% to about 3.0%, including from about 0.02% to about2.0%.

In other embodiment, the HMO mixture of this invention can be comprisedin a pharmaceutical composition. The pharmaceutical composition cancontain a pharmaceutically acceptable carrier, e.g. phosphate bufferedsaline solution, mixtures of ethanol in water, water and emulsions suchas an oil/water or water/oil emulsion, as well as various wetting agentsor excipients. The pharmaceutical composition can also contain othermaterials that do not produce an adverse, allergic or otherwise unwantedreaction when administered to non-infants. The carriers and othermaterials can include solvents, dispersants, coatings, absorptionpromoting agents, controlled release agents, and one or more inertexcipients, such as starches, polyols, granulating agents,microcrystalline cellulose, diluents, lubricants, binders, anddisintegrating agents. If desired, tablet dosages of the anti-infectivecompositions can be coated by standard aqueous or non-aqueoustechniques.

The pharmaceutical compositions can be administered orally, e.g. as atablet, capsule, or pellet containing a predetermined amount, or as apowder or granules containing a predetermined concentration or a gel,paste, solution, suspension, emulsion, syrup, bolus, electuary, orslurry, in an aqueous or non-aqueous liquid, containing a predeterminedconcentration. Orally administered compositions can include binders,lubricants, inert diluents, flavouring agents, and humectants. Orallyadministered compositions such as tablets can optionally be coated andcan be formulated so as to provide sustained, delayed or controlledrelease of the mixture therein.

The pharmaceutical compositions can also be administered by rectalsuppository, aerosol tube, naso-gastric tube or direct infusion into theGI tract or stomach.

The pharmaceutical compositions can also include therapeutic agents suchas antiviral agents, antibiotics, probiotics, analgesics, andanti-inflammatory agents. The proper dosage of these compositions for anon-infant human can be determined in a conventional manner, based uponfactors such immune status, body weight and age. In some cases, thedosage will be at a concentration similar to that found for the HMOs inhuman breast milk. The required amount would generally be in the rangefrom about 200 mg to about 20 g per day, in certain embodiments fromabout 300 mg to about 15 g per day, from about 400 mg to about 10 g perday, in certain embodiments from about 500 mg to about 10 g per day, incertain embodiments from about 1 g to about 10 g per day. Appropriatedose regimes can be determined by conventional methods.

Pharmaceutical compositions of the invention can be used for treatmentof a concerned disease in combination with other medicaments prescribedfor said disease, e.g. in combination with an anti-diabetes medicine orantibiotic therapy.

Yet in other embodiment, the composition comprising the HMO mixture ofthis invention is a unit dosage form. The unit dosage form can containan acceptable carrier, e.g. phosphate buffered saline solution, mixturesof ethanol in water, water and emulsions such as an oil/water orwater/oil emulsion, as well as various wetting agents or excipients. Theunit dosage form can also contain other materials that do not produce anadverse, allergic or otherwise unwanted reaction when administered to apatient. The carriers and other materials can include solvents,dispersants, coatings, absorption promoting agents, controlled releaseagents, and one or more inert excipients, such as starches, polyols,granulating agents, microcrystalline cellulose, diluents, lubricants,binders, and disintegrating agents. If desired, tablet dosages of thecomposition can be coated by standard aqueous or nonaqueous techniques.

A unit dosage form of this invention can be administered orally, e.g. asa tablet, capsule, or pellet containing a predetermined amount of themixture, or as a powder or granules containing a predeterminedconcentration of the mixture or a gel, paste, solution, suspension,emulsion, syrup, bolus, electuary, or slurry, in an aqueous ornonaqueous liquid, containing a predetermined concentration of themixture. An orally administered composition can include one or morebinders, lubricants, inert diluents, flavouring agents, and humectants.An orally administered composition such as a tablet can optionally becoated and can be formulated so as to provide sustained, delayed orcontrolled release of the HMO mixture therein.

A unit dosage form of this invention can also be administered by rectalsuppository, aerosol tube, naso-gastric tube or direct infusion into theGI tract or stomach.

A unit dosage form of this invention can also include therapeutic agentssuch as antiviral agents, antibiotics, probiotics, analgesics, andanti-inflammatory agents. The proper dosage of such a composition for apatient can be determined in a conventional manner, based upon factorssuch as the patient's immune status, body weight and age. In some cases,the dosage will be at a concentration similar to that found for the HMOsof the composition in human breast milk. The required amount wouldgenerally be in the range from about 200 mg to about 20 g per day, incertain embodiments from about 300 mg to about 15 g per day, from about400 mg to about 10 g per day, in certain embodiments from about 500 mgto about 10 g per day, in certain embodiments from about 1 g to about 10g per day. Appropriate dose regimes can be determined by methods knownto those skilled in the art.

EXAMPLES Example 1

A total of 50 healthy male and female subjects are recruited toparticipate in the study. After a screening visit and run-in period of1-2 weeks, the subjects are selected and randomized into 2 groups, eachof 25 subjects. One group is administered a treatment product containing5 g of the following HMO mixture of this invention:

-   -   i) 14.2 wt % of LNT    -   ii) 5.3 wt % of LNnT    -   iii) 63.7 wt % of 2′-FL    -   iv) 4.2 wt % of DFL,    -   v) 3.7 wt % of 3′-SL and    -   vi) 8.9 wt % of 6′-SL

The other group is administered a placebo (containing 2 grams ofglucose). The treatment product and the placebo are in powder form in aunit dosage container.

The subjects are eligible to participate if they are at least 18 yearsof age. All recruited subjects are able and willing to understand andcomply with the study procedures. Subjects are excluded if: they haveparticipated in a clinical study one month prior to screening visit;they have abnormal results in the screening tests which are clinicallyrelevant for study participation; they are suffering for a severedisease such as malignancy, diabetes, severe coronary disease, kidneydisease, neurological disease, or severe psychiatric disease or anycondition which can confound the results of the study; used highly dosedprobiotic supplements (yoghurt allowed) for 3 months prior to the study;consumed antibiotic drugs 3 months prior to the study; consumed on aregular basis any medication that might interfere with symptomevaluation 2 weeks prior to the study; and pregnant or lactating.

At the screening visit, medical history and concomitant medication isregistered and a blood sample for safety analyses is collected. A faecalsample kit is distributed. Subjects are instructed to keep their samplesin the freezer until the next visit.

At the second visit, eligibility criteria are checked and eligiblesubjects are randomised to the two arms in the trial. The faecal samplesare collected and equipment for new samples are distributed. Subjectsare familiarised with an interactive internet enabled system whichrecords data daily and are provided with either treatment or placeboproducts. Subjects are reminded not to change their usual diet duringthe study. Blood samples are collected for biomarker studies. The faecalsamples are stored at −80° C. until analysis. Faecal samples aresubjected to 16S rRNA sequencing analysis.

The study runs for 8 weeks with the subjects consuming either a placeboor a treatment product daily. Subjects are instructed to consume theproducts in the morning with breakfast. Compliance is monitored throughthe interactive internet enabled system. The subjects also use thesystem to record:

-   -   Bristol Stool Form (BSF) scale information,    -   symptom information such as abdominal pain, abdominal        discomfort, abdominal cramping, abdominal bloating, and        abdominal fullness,    -   additional Gastrointestinal Symptom Rating Scale (GSRS)        information.

This questionnaire includes 15 items covering five dimensions (abdominalpain, indigestion, reflux, diarrhoea, constipation) and uses aseven-graded Likert scale.

At the end of the study, each subject has an exit visit with the medicalteam. Faecal samples and blood samples are collected and analysed asbefore.

The faecal analysis indicates that the subjects treated with the HMOmixture of this invention have increased abundance of Bifidobacteriumand Barnesiella and reduced abundance of Firmicutes, especiallyClostridia, and Ruminococcus gnavus. The abundance of Faecalibacteriumis unchanged in these subjects. The abundance of Proteobacteria isdecreased in these subjects.

Example 2

A total of 50 healthy male and female subjects are recruited toparticipate in the study. After a screening visit and run-in period of1-2 weeks, the subjects are selected and randomized into 2 groups, eachof 25 subjects. One group is administered a treatment product containing5 g of the following HMO mixture of this invention:

-   -   i) 14.2 wt % of LNT    -   ii) 5.3 wt % of LNnT    -   iii) 63.7 wt % of 2′-FL    -   iv) 4.2 wt % of 3-FL,    -   v) 3.7 wt % of 3′-SL and    -   vi) 8.9 wt % of 6′-SL

The other group is administered a placebo (containing 2 grams ofglucose). The treatment product and the placebo are in powder form in aunit dosage container.

The subjects are eligible to participate if they are at least 18 yearsof age. All recruited subjects are able and willing to understand andcomply with the study procedures. Subjects are excluded if: they haveparticipated in a clinical study one month prior to screening visit;they have abnormal results in the screening tests which are clinicallyrelevant for study participation; they are suffering for a severedisease such as malignancy, diabetes, severe coronary disease, kidneydisease, neurological disease, or severe psychiatric disease or anycondition which can confound the results of the study; used highly dosedprobiotic supplements (yoghurt allowed) for 3 months prior to the study;consumed antibiotic drugs 3 months prior to the study; consumed on aregular basis any medication that might interfere with symptomevaluation 2 weeks prior to the study; and pregnant or lactating.

At the screening visit, medical history and concomitant medication isregistered and a blood sample for safety analyses is collected. A faecalsample kit is distributed. Subjects are instructed to keep their samplesin the freezer until the next visit.

At the second visit, eligibility criteria are checked and eligiblesubjects are randomised to the two arms in the trial. The faecal samplesare collected and equipment for new samples are distributed. Subjectsare familiarised with an interactive internet enabled system whichrecords data daily and are provided with either treatment or controlproducts. Subjects are reminded not to change their usual diet duringthe study. Blood samples are collected for biomarker studies. The faecalsamples are stored at −80° C. until analysis. Faecal samples aresubjected to 16S rRNA sequencing analysis.

The study runs for 8 weeks with the subjects consuming either a placeboor a treatment product daily. Subjects are instructed to consume theproducts in the morning with breakfast. Compliance is monitored throughthe interactive internet enabled system. The subjects also use thesystem to record:

-   -   Bristol Stool Form (BSF) scale information,    -   symptom information such as abdominal pain, abdominal        discomfort, abdominal cramping, abdominal bloating, and        abdominal fullness,    -   additional Gastrointestinal Symptom Rating Scale (GSRS)        information.

This questionnaire includes 15 items covering five dimensions (abdominalpain, indigestion, reflux, diarrhoea, constipation) and uses aseven-graded Likert scale.

At the end of the study, each subject has an exit visit with the medicalteam. Faecal samples and blood samples are collected and analysed asbefore.

The faecal analysis indicates that the subjects treated with the HMOmixture of this invention have increased abundance of Bifidobacteriumand Barnesiella and reduced abundance of Firmicutes, especiallyClostridia, and Ruminococcus gnavus. The abundance of Faecalibacteriumis unchanged in these subjects. The abundance of Proteobacteria isdecreased in these subjects.

Example 3

Twenty 7-week-old C57BL/6J female mice are individually housed to avoidcontamination between mice and provided with irradiated food and water.The mice are separated into 2 groups, each of 10 mice.

The mice are treated with ampicillin (0.5 g/liter) in their drinkingwater, which is changed every 3 days. After 1 week, the ampicillinaddition to the drinking water is terminated. Thereafter, 1 group isadministered a treatment product containing the following HMO mixture ofthis invention:

-   -   14.2 wt % of LNT    -   5.3 wt % of LNnT    -   63.7 wt % of 2′-FL    -   4.2 wt % of DFL,    -   3.7 wt % of 3′-SL and    -   8.9 wt % of 6′-SL.

The treatment product is added to the drinking water of 1 group at atotal concentration of 40 mg/ml. The other group receives drinking waterwith 40 mg/ml of glucose. Fresh water is administered daily, and allmice have free access to the drinking water. The mice are fed a rodentchow and are given fresh chow daily.

Two days after termination of the ampicillin treatment, mice of eachgroup are infected by oral gavage with a vancomycin-resistantEnterococcus faecium strain (VRE). Fresh faecal pellets are collected atdifferent time points to determine the VRE levels. VRE is quantified byplating serial dilutions of faecal pellets on Enterococcosel agar plateswith vancomycin. VRE colonies are identified by appearance and confirmedby Gram staining. PCR of the vanA gene, which confers resistance tovancomycin, is used to confirm the presence of VRE in infected mice.

The mice are monitored for 2 weeks and are then euthanized. Luminalcontents from the ilium, cecum and colon are collected and immediatelyfrozen and stored at −80° C. DNA is extracted using a 96-well PowerSoilDNA Isolation Kit (MO-BIO). A minimum of one sample-well per plate iskept empty to serve as a negative control during PCR. PCR is done withthe forward primer S-D-Bact-0341-b-S-17 and reverse primerS-D-Bact-0785-a-A-21 (Klindworth et al. Nucleic Acids Res. 41, e1(2013)) with Illumina adapters attached. These are universal bacterial16S rDNA primers, which target the V3-V4 region. The following PCRprogram is used: 98° C. for 30 sec, 25× (98° C. for 10 s, 55° C. for 20s, 72° C. for 20 s), 72° C. for 5 min. Amplification is verified byrunning the products on a 1% agarose gel. Barcodes are added in a nestedPCR using the Nextera Index Kit V2 (Illumina) with the following PCRprogram: 98° C. for 30 sec, 8× (98° C. for 10 s, 55° C. for 20 s, 72° C.for 20 s), 72° C. for 5 min. Attachment of primers is verified byrunning the products on a 1% agarose gel.

Products from the nested PCR are normalized using the SequalPrepNormalization Plate Kit and pooled. Pooled libraries are concentrated byevaporation and the DNA concentration of pooled libraries wisas measuredon a Qubit fluorometer using the Qubit High Sensitivity Assay Kit(Thermo Fisher Scientific). Sequencing is done on a MiSeq desktopsequencer using the MiSeq Reagent Kit V3 (Illumina) for 2×300 bppaired-end sequencing. The 64-bit version of USEARCH (Edgar, 2013) isused for bioinformatical analysis of the sequence data.

In the mice treated with the HMO mixture of this invention, VREcolonisation is reduced to undetectable levels within 14 days. Thedensity of VRE reduces within 5 days. The mice treated with the HMOmixture also showed a higher abundance of Porphyromonadaceae, especiallyBarnesiella. The untreated mice continue to harbour large numbers of VREthroughout the intestine.

Example 4

Twenty 7-week-old C57BL/6J female mice are individually housed to avoidcontamination between mice and provided with irradiated food and water.The mice are separated into 2 groups, each of 10 mice.

The mice are treated with ampicillin (0.5 g/liter) in their drinkingwater, which is changed every 3 days. After 1 week, the ampicillinaddition to the drinking water is terminated. Thereafter, 1 group isadministered a treatment product containing the following HMO mixture ofthis invention:

-   -   14.2 wt % of LNT    -   5.3 wt % of LNnT    -   63.7 wt % of 2′-FL    -   4.2 wt % of 3-FL,    -   3.7 wt % of 3′-SL and    -   8.9 wt % of 6′-SL.

The treatment product is added to the drinking water of 1 group at atotal concentration of 40 mg/ml. The other group receives drinking waterwith 40 mg/ml of glucose. Fresh water is administered daily, and allmice have free access to the drinking water. The mice are fed a rodentchow and are given fresh chow daily.

Two days after termination of the ampicillin treatment, mice of eachgroup are infected by oral gavage with a vancomycin-resistantEnterococcus faecium strain (VRE). Fresh faecal pellets are collected atdifferent time points to determine the VRE levels. VRE is quantified byplating serial dilutions of faecal pellets on Enterococcosel agar plateswith vancomycin. VRE colonies are identified by appearance and confirmedby Gram staining. PCR of the vanA gene, which confers resistance tovancomycin, is used to confirm the presence of VRE in infected mice.

The mice are monitored for 2 weeks and are then euthanized. Luminalcontents from the ilium, cecum and colon are collected and immediatelyfrozen and stored at −80° C. DNA is extracted using a 96-well PowerSoilDNA Isolation Kit (MO-BIO). A minimum of one sample-well per plate iskept empty to serve as a negative control during PCR. PCR is done withthe forward primer S-D-Bact-0341-b-S-17 and reverse primerS-D-Bact-0785-a-A-21 (Klindworth et al. Nucleic Acids Res. 41, e1(2013)) with Illumina adapters attached. These are universal bacterial16S rDNA primers, which target the V3-V4 region. The following PCRprogram is used: 98° C. for 30 sec, 25× (98° C. for 10 s, 55° C. for 20s, 72° C. for 20 s), 72° C. for 5 min. Amplification is verified byrunning the products on a 1% agarose gel. Barcodes are added in a nestedPCR using the Nextera Index Kit V2 (Illumina) with the following PCRprogram: 98° C. for 30 sec, 8× (98° C. for 10 s, 55° C. for 20 s, 72° C.for 20 s), 72° C. for 5 min. Attachment of primers is verified byrunning the products on a 1% agarose gel.

Products from the nested PCR are normalized using the SequalPrepNormalization Plate Kit and pooled. Pooled libraries are concentrated byevaporation and the DNA concentration of pooled libraries wisas measuredon a Qubit fluorometer using the Qubit High Sensitivity Assay Kit(Thermo Fisher Scientific). Sequencing is done on a MiSeq desktopsequencer using the MiSeq Reagent Kit V3 (Illumina) for 2×300 bppaired-end sequencing. The 64-bit version of USEARCH (Edgar, 2013) isused for bioinformatical analysis of the sequence data.

In the mice treated with the HMO mixture of this invention, VREcolonisation is reduced to undetectable levels within 14 days. Thedensity of VRE reduces within 5 days. The mice treated with the HMOmixture also showed a higher abundance of Porphyromonadaceae, especiallyBarnesiella. The untreated mice continue to harbour large numbers of VREthroughout the intestine.

Example 5

Forty-five 6-week-old female C57BL/6 mice are injected subcutaneouslywith B16 melanoma cells. The mice are then randomly assigned to threegroups (fifteen mice in each group), and individually housed to avoidcontamination between mice. The mice are fed a pelleted semi-syntheticAIN 76 diet (Research Diets Inc., New Brunswick, N.J.) with: 5% of thefollowing HMO mixture of this invention in the feed for group A:

-   -   14.2 wt % of LNT    -   5.3 wt % of LNnT    -   63.7 wt % of 2′-FL    -   4.2 wt % of DFL,    -   3.7 wt % of 3′-SL and    -   8.9 wt % of 6′-SL;        5% of the following HMO mixture of this invention in the feed        for group B:    -   14.2 wt % of LNT    -   5.3 wt % of LNnT    -   63.7 wt % of 2′-FL    -   4.2 wt % of 3-FL,    -   3.7 wt % of 3′-SL and    -   8.9 wt % of 6′-SL; and        5% corn-starch in the feed for group C (control).

Fresh water is administered daily and all mice have free access todrinking water and feed. Three days after the tumor implantation, miceare injected with 200 μg of CTLA-4 antibodies. Additional antibodytreatments are given every 3 days until the end of the experiment (14days). Food consumption, bodyweight and tumor volume (diameter) aremeasured throughout the study. Fresh faecal samples are collected at day0, 4, 7, 10, and 14. Samples are immediately frozen and stored at −80°C. until further analysis.

Fourteen days after implantation of the B16 cells, mice are euthanizedby cervical dislocation. Tumors are excised and weighed. Sections oftumors are fixed in 4% paraformaldehyde and stored at −80° C. Mucosaland intestinal contents from caecum and colon are removed and stored at−80° C.

For analysis of tumor infiltrating T cells, tumors are minced into smallpieces and digested with 0.2 mg/ml DNase and 1.67 Wunsch U/ml Liberase(Roche). Obtained cell suspensions are filtered through a 40-μm nyloncell strainer and red blood cells lysed. CD8⁺ T cells are purified fromB16 tumor-derived cell suspensions using CD8α (Ly-2) MicroBeads(Miltenyi Biotec), following the manufacturer's protocol. Obtained cellsare incubated for 2 hours in the presence of 1 μl/ml of brefeldin A,washed and incubated with rat anti-mouse CD16/CD32 mAb (2.4G2) to blocknonspecific binding, and then stained with CD8α-PE-Cy5 and CD69-PE,followed by intracellular staining with IFN-γ-PE-Cy7 or isotype controlantibodies according to the manufacturer's instructions (BDBiosciences—Pharmingen). The density of CD8 GFP⁺ and GFP⁻ T cells in thetumors are calculated by dividing the total number of obtained cells bythe tumor weight.

To assess the microbiota profile, DNA is extracted from the faecalsamples, mucosal and intestinal content using a 96-well PowerSoil DNAIsolation Kit (MO-BIO). A minimum of one sample-well per plate is keptempty to serve as a negative control during PCR. PCR is done with theforward primer S-D-Bact-0341-b-S-17 and reverse primerS-D-Bact-0785-a-A-21 (Klindworth et al. Nucleic Acids Res. 41, e1(2013)) with Illumina adapters attached. These are universal bacterial16S rDNA primers, which target the V3-V4 region. The following PCRprogram is used: 98° C. for 30 sec, 25× (98° C. for 10 s, 55° C. for 20s, 72° C. for 20 s), 72° C. for 5 min. Amplification is verified byrunning the products on a 1% agarose gel. Barcodes are added in a nestedPCR using the Nextera Index Kit V2 (Illumina) with the following PCRprogram: 98° C. for 30 sec, 8× (98° C. for 10 s, 55° C. for 20 s, 72° C.for 20 s), 72° C. for 5 min. Attachment of primers is verified byrunning the products on a 1% agarose gel. Products from the nested PCRare normalized using the SequalPrep Normalization Plate Kit and pooled.Pooled libraries are concentrated by evaporation and the DNAconcentration of pooled libraries is measured on a Qubit fluorometerusing the Qubit High Sensitivity Assay Kit (Thermo Fisher Scientific).Sequencing is done on a MiSeq desktop sequencer using the MiSeq ReagentKit V3 (Illumina) for 2×300 bp paired-end sequencing. The 64-bit versionof USEARCH (Edgar, 2013) is used for bioinformatical analysis of thesequence data.

The results show that ingestion of both HMO mixtures beneficiallymodulates the microbiota by increasing the abundance of bifidobacteriaboth in the mucosal and luminal intestinal environment. Additionally,the results show that the abundance of bifidobacteria positivelycorrelates with the priming and accumulation of infiltrating T cells inthe tumor microenvironment and negatively correlates with tumor size.Collectively, these results indicate that both HMO mixtures can increasethe abundance of bifidobacteria and through this increase the efficacyof anti-cancer agents against tumor outgrowth.

Example 6

Mice (5 weeks old; n=30) are randomly allocated to one of the followinggroups: A) control (n=10); B) 1% mixture of HMOs (n=10); C) 5% mixtureof HMOs (n=10). The composition of the HMO blend is given in the tablebelow.

g/100 ml 1% 5% 2′-FL (g) 0.64 3.20 LNnT (g) 0.05 0.25 LNT (g) 0.14 0.703′-SL (g) 0.05 0.25 6′-SL (g) 0.09 0.45 DFL (g) 0.05 0.25

The HMOs are provided in drinking water. All animals have free access tosame diet (KLIBA 2122).

After 2 weeks, the mice are challenged with Influenza strain PR8 at adose of 100 PFU per mouse by intranasal inoculation. The mice aremonitored for the next 14 days to assess clinical score of illnesssymptoms and body weight loss.

The results show that the HMO mixture protects against sickness symptomsand body weight loss caused by the influenza virus infection.

The invention claimed is:
 1. A method of treating a viral infection in anon-infant human, comprising: administering to the non-infant human asynthetic mixture of human milk oligosaccharides (HMOs) consistingessentially of lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT),2′-O-fucosyllactose (2′-FL), 3′-O-sialyllactose (3′-SL),6′-O-sialyllactose (6′-SL), either 3-fucosyllactose (3-FL) ordifucosyllactose (DFL), and optionally lactose.
 2. The method of claim1, wherein the synthetic mixture consists essentially of LNnT, LNT,2′-FL, 3′-SL, 6′-SL, DFL and, optionally, lactose.
 3. The method ofclaim 2, wherein the synthetic mixture consists essentially of i. about55 wt % to about 75 wt % of 2′-FL; ii. about 2 wt % to about 10 wt % ofLNnT; iii. about 10 wt % to about 20 wt % of LNT; iv. about 1 wt % toabout 15 wt % of DFL; v. about 1 wt % to about 10 wt % of 3′-SL; and vi.about 1 wt % to about 15 wt % of 6′-SL.
 4. The method of claim 1,wherein the synthetic mixture consists essentially of i. about 55 wt %to about 75 wt % of 2′-FL; ii. about 2 wt % to about 10 wt % of LNnT;iii. about 10 wt % to about 20 wt % of LNT; iv. about 1 wt % to about 15wt % of 3-FL; v. about 1 wt % to about 10 wt % of 3′-SL; and vi. about 1wt % to about 15 wt % of 6′-SL.
 5. The method of claim 4, wherein thesynthetic mixture consists essentially of i. about 60 wt % to about 70wt % of 2′-FL; ii. about 3 wt % to about 7 wt % of LNnT; iii. about 12wt % to about 18 wt % of LNT; iv. about 2 wt % to about 10 wt % of 3-FL;v. about 2 wt % to about 8 wt % of 3′-SL; and vi. about 5 wt % to about15 wt % of 6′-SL.
 6. The method of claim 1, wherein the syntheticmixture is a component of a nutritional or a pharmaceutical composition.7. The method of claim 1, wherein the viral infection is influenza.